Purification Fold Research Articles

EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LIPASE FROM THE DIGESTIVE DUCT OF COMMON CARP CYPRINUS CARPIO L.

This study was aimed to use digestive duct of common carp Cyprinus Carpio L. as economic source for fish lipase extraction . The enzyme was extracted using potassium phosphate buffer (pH 7 , 0.2 M ). The crude extract then precipitated at the saturation range between 40 – 80 % and dialyzed . Further purified carried out through ion exchange chromatography using DEAE-Cellulose column (2.5 × 30 cm) and gel filtration chromatography by Sephadex G-100 column (1.5 x 60 cm). The purified lipase had a molecular weight of 37.150KDa ,and optimum pH and temperature for enzyme activity about 45◦C and 6.5 using olive oil as substrate .The specific activity, number of purification folds and yield of purified Lipase were 146.48U/mg, 9.76 and 40.50% respectively.

ASSESSEMENT OF PURIFIED COLLAGENASE INHIBITION ACTIVITY FROM STAPHYLLOCOCCUS AUREUS HN77 BY SOME LOCAL PLANTS EXTRACT

This study was aimed to inhibition of purified collagenase produced from S. aureus by local plants inhibitors (fruits and leaves). Sixty two local isolates of S. aureus which were isolated and identified by biochemical test subjected to the primary and secondary screening processes to select the active S. aureus isolate for collagenase production. Among these isolates, forty nine isolates with maximum zone of hydrolysis ability in primary screening (the ratio of Z/G more than 10 mm) were selected for secondary screening. It has been found that S. aureus HN77 had the highest productivity of the enzyme (49.122 U/mg protein). The selected isolate with highest level of collagenase activity was identified as S. aureus HN77 according to vitek test. The optimum conditions of collagenase production by selected isolate using submerged fermentation were obtained with medium (D) contain collagen as the best production medium, 2% starch as the best carbon source, 2% yeast extract/peptone as nitrogen source, temperature 37 °C and pH 7, after 48 hr. of incubation period, the specific activity was reached to1502.15 U/mg. The enzyme was purified by gel filtration chromatography using Sephadex G-150 then concentration by sucrose. The results shown an increase in final purification folds 6.1 time with an enzyme yield of 85.2%.The purified enzyme was exhibited maximal activity and stability at pH 7.0. The optimal temperature for purified enzyme activity was 45°C and it was stable until 45°C.The highest rate of enzyme specificity found with collagen. Collagenase was inhibited with local plants extract (fruit and leaves). Fruit of black fig extract inhibit 90% of collagenase activity while the inhibition efficiency of collagenase with grape leaf was reached to 12%.

Characterization of a New Thermostable Carboxylesterase from Aneurinibacillus sp. PDF24

In this study, esterase of Aneurinibacillus sp. PDF24 strain, a thermophilic bacteria, was purified to homogenity (5.25 fold purification) by column chromotography, and characterized. The molecular weight of Aneurinibacillus sp. PDF24 esterase was determined about 40 kDa. The maximum activity of the purified esterase was analyzed at 55°C, pH 8.5. The esterase was found to be stable at 40ºC, 50ºC and 60ºC for 1 hour. Km and Vmax values for p-nitrophenyl butyrate were determined as 0.120 mM and 3164.8 U/mg, respectively. Considering Km values in the literature, Aneurinibacillus sp. PDF24 esterase was found to have a good Km value compared to other esterases. In the presence of 1 mM and 5 mM metal salts of Mg2+, Li+, Ca2+, K+, no significant change occured in enzyme activity. The activity of Aneurinibacillus sp PDF24 esterase was found to be stable also in the presence of ethanol, DMSO, EDTA, DTT and ß-mercaptoethanol. The data obtained suggest that the enzyme is a serine esterase, not a metalloprotein, and that disulfide bonds are not required to maintain enzyme conformation, and therefore, depending on its features, this esterase may be a suitable candidate for industrial applications.

Lactoperoxidase partitioning from whey using the reverse micelles of non‐ionic/ionic mixed surfactants: Improvement of back extraction

AbstractThe reverse micellar system formed with a mixture of ionic and non‐ionic surfactants, AOT (Sodium bis[2‐ethyl hexyl] sulfosuccinate)/Tween 80 in isooctane, was studied for the selective extraction of Bovine Lactoperoxidase (LP) from acid whey. The effect of pH and ionic strength of acid whey and surfactant concentrations were studied and a maximum of 86% LP was extracted from acid whey at pH 9.5 with the addition of 0.2 M KCl to the reverse micelles formed with AOT (115 mM)/Tween 80 (23 mM). The back extraction of LP was studied at different aqueous stripping phase pH, ionic strength and concentration of counter‐ionic surfactant Cetyltrimethylammonium bromide (CTAB). The back extraction of 112% with 80% LP recovery was achieved when the stripping phase pH was 10.5 with 1.5 M KCl and 60 mM CTAB. The antimicrobial activity of the extracted LP showed reduction in colony‐forming units of S. aureus.Novelty impact statementThe reverse micelles formed with AOT/Tween 80 surfactant mixture minimize the pH‐dependent denaturation of LP and widen the pH window (7.5–9.5) for LP extraction. The back extraction of LP from the reverse micellar phase to aqueous stripping phase was improved by the addition of CTAB as counter‐ionic surfactant. A purification fold of 11.26 achieved with minimal loss in activity of LP by retaining the native Antimicrobial characteristic.

Isolation, purification and partial characterization of ficin from Ficus carica latex

This study aimed at the isolation, purification and characterization of fig latex ficin. Samples were collected from fruit joint with the fig tree. Supernatant was decanted from fig latex after centrifugation at 3000 rpm which represents crude enzyme extract. Latex content was concentrated by sucrose powder. Purification steps were carried out with ion-exchange and gel filtration using DEAE- cellulose and sephadex G-50 columns respectively. Enzyme yield and purification folds were 51.688% and 15.635 respectively with specific activity 148.336 U/mg. By using gel filtration, molecular weight of the purified enzyme was estimated to be 22.725 Da. The pH at which ficin showing activity ranged between 6-8.5. Ficin showed a good stability in pH ranging between 6-8. Optimum temperature for activity and stability were 50 C○ and 25-40 C○ respectively. Activation energy for substrate conversion of this enzyme was calculated to be 10.22 Kca/mol.

Biosynthesis of gallic acid and tannase from Dacryodes edulis (African Pear) seeds using Aspergillus niger SCSGAF0145 by submerged fermentation

Aspergillus niger SCSGAF0145 previously isolated and identified by our group as a tannase producer from soil was grown under submerged fermentation using pear seed as source of tannic acid. Crude tannase was precipitated out of the cell free broth and purified by gel filtration gave a purified enzyme with specific activity of 6.65 U/mg, with a final yield and purification fold of 42.08% and 5.94 respectively. The deduced Vmax of the purified tannase was 17.57 U/mL and Km was 1.59 mg/mL. The optimal temperature and pH of the enzyme using tannic acid as sole carbon source was 40 °C and 5.5 respectively. Conclusively, pear seed is a good substrate for gallic acid and tannase production by Aspergillus niger SCSGAF0145 and tannase secreted can be explored in various industrial processes.

Purification and Partial Characterization of Tannase produced by Aspergillus niger SCSGAF0145 using Arachis hypogaea (Groundnut) Shell

Tannase have been widely secreted industrially by fungi using tannin rich agricultural waste as substrate. Aspergillus niger SCSGAF0145, was used for the secretion of tannase using groundnut shell as substrate under submerged fermentation. The crude tannase secreted was precipitated out using ammonium sulphate and purified on Sephadex G-150. Kinetic parameters of the secreted tannase were determined using tannic acid as substrate. The estimated biomass weight, tannin concentration, gallic acid concentration and tannase activity of the crude enzyme after fermentation was 81.00±0.08 mg, 1.31±0.01 mg/mL, 0.67±0.03 µg/mL and 6.04 U/mL respectively. The purification fold and enzyme yield after purification were 13.44 and 47.86%. The purified enzyme showed maximal activity at a pH of 5.5 and temperature of 35oC while producing Km and Vmax of 0.43 moles/L and Vmax is 21.55 U/mL respectively using tannic acid as its sole carbon source. In conclusion, Aspergillus niger SCSGAF0145 secreted tannase of high activity using groundnut shell as source of tannic acid which could be utilized for various industrial purposes.

Bioprocessing and purification of extracellular L-asparaginase produced by endophytic Colletotrichum gloeosporioides and its anticancer activity

L-asparaginase is an enzyme commonly used to treat acute lymphoblastic leukemia. Commercialized bacterial L-asparaginase has been reported to cause several life-threatening complications during treatment, hence the need to seek alternative sources of L-asparaginase. In this study, the novelty of upstream and downstream bioprocessing of L-asparaginase from a fungal endophyte, Colletotrichum gloeosporioides, and the cytotoxicity evaluation was demonstrated. Six variables (carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate) known to influence L-asparaginase production were studied using One-Factor-At-A-Time (OFAT) approach, with four significant variables further optimized using Response Surface Methodology (RSM). The crude extract produced using optimized condition was purified, characterized and examined for its anticancer effect. Purification of fungal L-asparaginase was performed via ultrafiltration and size exclusion chromatography, which are less common techniques. The protein profile and monomeric weight of L-asparaginase were determined using SDS-PAGE and Western blot. Cytotoxicity of purified L-asparaginase on leukemic Jurkat E6 and oral carcinoma cells were studied using MTS assay for 24 h and 48 h. OFAT results from optimization showed that glucose and L-asparagine concentrations, incubation period and temperature, were significant factors affecting L-asparaginase production by C. gloeosporioides. RSM analysis further evidence the significant interaction between glucose and L-asparagine concentrations in inducing L-asparaginase production. Purified L-asparaginase was profiled with specific activity of 255.02 IU/mg protein, purification fold of 6.12, and 34.63% of enzyme recovery. SDS and Western blot revealed that the purified L-asparaginase might be a tetramer with monomeric units of 25 kDa. Purified L-asparaginase was discovered to be more efficient against Jurkat leukemic cells than against H103 oral carcinoma cells, as lower IC50 value was observed for Jurkat cell lines (46 .36 ± 1.52 µg/mL for Jurkat and 125.56 ± 7.28 µg/mL for H103). In short, purified L-asparaginase derived from endophytic C. gloeosporioides showed high purity and significant anticancer effect toward cancer cells. This study therefore demonstrated the potential of fungal L-asparaginase as alternative chemotherapy drug in the future.

Characterization of a novel end product tolerant and thermostable cellulase from Neobacillus sedimentimangrovi UE25

Recent advancements in biorefinery processes necessitate search for cost effective and thermostable cellulases. This study was designed to characterize the cellulase obtained from a thermophilic bacterium, Neobacillus sedimentimangrovi UE25. A combined pretreatment of NaOH and methyltrioctylammonium chloride was given to sugarcane bagasse (SB) for lignin removal and the pretreated SB was utilized as a carbon source for the cellulase production. The thermostable cellulase thus obtained was characterized by adopting central composite design which has not been reported earlier for this purpose. Cellulase showed its maximum activity at pH 7 and temperature 60 ℃ and it remained active in the presence of many salts and detergents. Endoglucanase (EG) was found to be stable for 30 min at 80 ℃. The purification of EG by using DEAE column yielded specific activity and purification fold of 365.866 IU mg−1 and 4.264, respectively. The purified EG had a molecular weight of ∼45 kDa. End product tolerance of EG was also evident, as an activity of 228.57 IU mL−1 was observed in the presence of 60 mM glucose which revealed that it does not lose its activity upon accumulation of end-product when the reaction is prolonged. The purified EG exhibited Vmax and Km of 294 U mL−1 min−1 and 36 µM, respectively, in the presence of 60 mM glucose. This novel thermostable cellulase can finds its applications in industrial sector.

Purification and characterization of glutaminase from Iraqi fruit of ‎ Capsicum annuum

L-glutaminase has recently attracted much attention due to its medicinal and industrial potential. It's an antileukemic drug with flavor-enhancing properties when used in fermented foods manufacturing. The enzyme purification was determined by ammonium sulfate precipitation, Ion exchange chromatography and gel filtration chromatography with estimating glutaminase activity and characterization of purified glutaminase in several parameters such as substrate concentration and reaction time pH, and temperature influence in this study. As a result, glutaminase is purified in three steps: ammonium sulfate precipitation with 30% saturation, DEAE-Cellulose and Sephacryl S-200. The specific activity increased to 85 U/mg protein with 4 folds of purification and 18% enzyme recovery. When glutaminase was incubated in the presence of 150 millimolar glutamine at thirty-five centigrade for thirty minutes, the enzyme reached its maximal activity of 1.8 u/ml, in the presence of a 0.05 Molar potassium phosphate buffer solution, at pH 8. Keywords: Capsicum annum, glutaminase, enzyme activity, glutaminase purification

Evaluation of the effect of Castanea sativa extracts on lipoxygenase activity

Lipoxygenase LOX is a lipolysis enzyme that oxidises unsaturated fatty acids like arachidonic and linoleic acids to form unhealthy chemicals such as dienes, leukotrienes and malondialdehyde in advanced oxidation, which is harmful to cells. In this study, the polyphenols and saponins compounds were extracted from Castanea sativa using Soxhlet for 3 days. Colourimetric and HPLC techniques were used to identify polyphenols and saponins in the extracts respectively. The effects of these extracts were evaluated on LOX activity, which was purified from the liver of Iraqi Cows (AL-Sharabi Cows) as well as on E. coli and Pseudomonas bacteria resistance. According to the LOX purification procedures, the specific activity increased from 0.001 to 0.03 U/mg with a purification fold of 30 times and a yield percentage of 174. Gallic acid 7.72 mg, Rutin 29.25 mg, Quercetin 27.6 mg, kaempferol 34.42 mg, Apigenin 5.25 mg and Catechin 25.8 mg/100 gm of Castanea fruit was obtained. The inhibitory effect of polyphenol and saponin extracts on LOX activity was at 100 and 40 µg respectively. Line weaver-Burk plot was used to investigate the type of inhibition that was found non-competitive. However, these extracts were studied at concentrations of 500, 250 and 125 µg on bacteria resistance. Polyphenols had the best effect at 500 and 125 µg on E. coli and Pseudomonas bacteria respectively. Whereas, saponin had the best effect at 250 and 125 µg for E. coli and 125 µg for Pseudomonas.

The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application.

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 μM mg protein−1 min−1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.

Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris.

β-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound β-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/μg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a Km value of 84.28 μM at pH 7.0 and synthesized β-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of β-1,3-glucan synthases in mushrooms.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and V max of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

Some properties of partially purified alpha amylase from Rice (Oryza sativa)

The present study was aimed at determining some properties of partially purified alpha amylase from rice (Oryza sativa). Different cultivars of rice were tested for alpha amylase production. The crude α-amylase was obtained by homogenization of day one germinated rice (cultivar ‘Election’) in sodium acetate buffer pH 4.0. After centrifugation, the crude enzyme was subjected to 20% (w/v) ammonia sulphate precipitation and dialysis, and its biochemical and kinetic properties were analyzed. After dialysis, the specific activity 1715.875 U/mg with purification fold of 3 and 60% recovery was obtained for the enzyme. The Km and Vmax were found to be 9 mg/ml and 178. µmol/min for rice starch, 5.150 mg/ml and 50.000 µmol/min for cassava starch, and 14.667 mg/ml and 111.111 µmol/min for maize starch, respectively. The specificity constant was found to be 13.888 min-1, 9.709 min-1 and 7.576 min-1 for rice, cassava and maize respectively. This shows that α-amylase has greater affinity for rice starch than the other starches used. The optimum pH and temperature were found to be 7.5 and 50 ºC respectively. The presence of metal ion such as Ca2+, Co2+, Fe3+, and Zn2+ activated α-amylase activity while Pb2+ and Mn2+ inhibited α-amylase activity. The results obtained from the present study indicated that Oryza sativa, is a good source of α-amylase for food industry, hence possess the properties for biotechnological applications.

Isolation, purification and biochemical characterization of a thermophilic alkaline protease from hot water spring bacteria

Search for a new alkaline and thermostable protease is crucial to explore their industrial applications. Therefore, a thermophilic, alkaliphilic bacteria producing serine alkaline protease was isolated from a hot water spring. Isolated bacteria were identified as B. licheniformis strain 9AkScKu3 as per the results revealed from biochemical studies and 16s rRNA gene sequencing analysis. The purification fold of the enzyme was 23.91 with yield of 18.54%. The purified protease was optimally active at pH 9 and a temperature of 50°C. The enzyme exhibited thermal stability up to 70ºC. Metal ions Ca2+, Mg2+ and Cu2+ enhanced the activity of enzyme. The enzyme exhibited compatibility with 3% surfactants showing more than 100% stability with SDS, tween 20 and triton-x100. Enzyme retained 100% activity with 5% (v/v) H2O2 and organic solvents like toluene and n-hexane at 10% (v/v) concentration. Thus isolated thermophilic alkaline protease is a promising enzyme applicable in detergent formulation.

Optimization of Aspergillus niger NRC1ami Pectinase Using Citrus Peel Pectin, Purification, and Thermodynamic Characterization of the Free and Modified Enzyme

Enzyme cost and stability are the main problems facing industrial applications. Consequently, Aspergillus nigerNRC1ami was isolated from rotten orange and recorded a promising pectinase activity (13.8 U/ml). The enzyme was optimized using citrus peel pectin as the sole carbon source and recorded (40 U/ml). It was purified by two steps purifications and recorded 632 purification folds. The pure enzyme showed 14.7% carbohydrate content and consists of 15 amino acids. Glutamic acid was the major (22%) followed by leucine (10.67%) and threonine was the minor (2.70%). A. niger NRC1ami pectinase was conjugated by covalent coupling to sodium periodate (NaIO4) activated polysaccharides. Galactomannan showed the highest recovered activity (85%) and 94.34% reduction in viscosity. The optimum temperature for the pure enzyme shifted from 40 to 45 °C after the conjugation process. Also, the free enzyme showed its optimum activity at pH 5 compared to pH 4, 5 in the conjugated form case. The thermal stability of the free enzyme greatly improved after the modification process. The conjugated process reduced the activation energy to 36%, prolonged the enzyme half-life 5.6-fold at 60 °C, and increase the deactivation energy (Ed) by about 19% in comparison to the free form. The D value of the conjugated enzyme increased to 13.2-fold at 50 °C compared to the free form. Gibbs's free energy (ΔG) of the enzyme increased after the modification process, while the enthalpy (ΔH) and entropy (ΔS) decreased. Na+ and Zn2+ had a stimulating effect on both enzyme forms.Graphical

Characterization & Partial purification of Microbial Lipase enzyme produce by Serratia nematodiphilia isolated from Paper & pulp effluent

As the demand is growing for paper and pulp at a highest peak, it not only imposes a severe threat on the forest resources but also on the environment. The effluent discharged by these paper and pulp industries is of great concern. Along with the biodegradation of theses effluents, we also aimed to produce microbial lipase. Lipases, a class of enzymes which catalyze the hydrolysis of long chain triglycerides. At present, microbial lipases are attaining much awareness with the rapid progress of enzyme technology. Lipases represent the most significant group of biocatalysts for industrial applications. In the present study various samples were collected from the paper and pulp effluent from Vapi, Gujarat. For preliminary understanding, total 18 bacterial isolates (JKB1 to JKB18) and 5 Fungi isolates (S1 to S5) were isolated out of that. Based on their primary screening technique, JKB1, JKB3, JKB5 and JKB21 bacterial isolates were studied for further for lipase production. Results show that isolate JKB3 was more potential and was studied further for characterization of lipase enzyme. It was identified as Serratia nematodaphilia. The results also indicate that the maximum activity was obtained i.e. 59.66 U/ml in optimized conditions like Glucose 2.75 U/ml, Tryptone 32.39 U/ml, Peanut oil 34.71U/ml, pH-7 64.92 U/ml, room temperature. At the end of partial purification, 5.15 fold purification of lipase was obtained. The current study represents the isolation and characterization of lipase enzyme which has a great applicability in paper & pulp effluent in deinking process in making of paper and increasing brightness, intensity and the pulping rate. It can also decrease chemical consumption, prolong machinery life, reduction in pollution, save energy, time and production cost.

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase from Burkholderia pseudomallei.

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC50 values of 1.53 and 18 IU, respectively.

An economical source for peroxidase: maize cobs

According to the current situation of peroxidase (POD), the relevant studies on this enzyme indicated its importance as a tool in clinical biochemistry and different industrial fields. Most of these studies used the fruits and vegetables as source of this enzyme. So that in order to couple the growing requirements for POD with the recent demands for reduc-ing disposal volume by recycling the plant waste, the aim of the present study was to extract POD through management of municipal bio-waste of Iraqi maize species. A simple, green and economical method was used to extract this enzyme. Our results revealed that maize cobs are rich sources of POD, where the activity of this enzyme was found to be 7035.54 U/g of cobs. In pilot experiments this enzyme was extracted from the cobs using an efficient extraction buffer with either Cetyl Trimethyl Ammonium Bromide (CTAB), or sonication. To purify the extracted enzyme the previous step was followed by aqueous two phase extraction (ATPE) using 20% (w/v) polyethylene glycol (PEG) and 9% (w/v) ammonium sulfate. The obtained results indicated that POD was partially purified with 2.36 fold of purification and 81.78% recovery. The optimum temperature and pH of the extracted POD activity as well as the enzyme thermal stabil-ity were determined and found to be 20°C, pH 6, and stable at 60°C for 10 minutes respectively. Out of the present study findings, it can be concluded that maize cobs are rich source for POD and the applied protocol could be poten-tially used for POD extraction with high level. Meantime, this study suggested a new strategy by which the environ-mental pollution results from accumulation of plant waste can be reduced.